Kinetic Evaluation of Human Cloned Coproporphyrinogen Oxidase Using a Ring Isomer of the Natural Substrate Marjorie A

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by ISU ReD: Research and eData

Illinois State University ISU ReD: Research and eData

Faculty Publications – Chemistry Chemistry

11-1-2005 Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate Marjorie A. Jones Illinois State University

Christopher L. Cooper Illinois State University

Timothy D. Lash Illinois State University

Follow this and additional works at: http://ir.library.illinoisstate.edu/fpchem Part of the Condensed Matter Physics Commons

Recommended Citation Jones, Marjorie A.; Cooper, Christopher L.; and Lash, Timothy D., "Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate" (2005). Faculty Publications – Chemistry. Paper 1. http://ir.library.illinoisstate.edu/fpchem/1

This Article is brought to you for free and open access by the Chemistry at ISU ReD: Research and eData. It has been accepted for inclusion in Faculty Publications – Chemistry by an authorized administrator of ISU ReD: Research and eData. For more information, please contact [email protected]. Illinois State University ISU ReD: Research and eData

Chemistry Department Arts and Sciences

11-1-2005 Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate Marjorie A. Jones Illinois State University

Christopher L. Cooper Illinois State University

Timothy D. Lash Illinois State University

Follow this and additional works at: http://ir.library.illinoisstate.edu/chem Part of the Chemistry Commons

Recommended Citation Jones, Marjorie A.; Cooper, Christopher L.; and Lash, Timothy D., "Kinetic evaluation of human cloned coproporphyrinogen oxidase using a ring isomer of the natural substrate" (2005). Chemistry Department. Paper 3. http://ir.library.illinoisstate.edu/chem/3

This Article is brought to you for free and open access by the Arts and Sciences at ISU ReD: Research and eData. It has been accepted for inclusion in Chemistry Department by an authorized administrator of ISU ReD: Research and eData. For more information, please contact [email protected]. © Med Sci Monit, 2005; 11(11): BR420-425 WWW.MEDSCIMONIT.COM PMID: 16258391 Basic Research

Received: 2005.06.28 Accepted: 2005.08.08 Kinetic evaluation of human cloned coproporphyrinogen Published: 2005.11.01 oxidase using a ring isomer of the natural substrate

Authors’ Contribution: Christopher L. CooperABCDEFG, Timothy D. LashABCDEFG, Marjorie A. JonesABCDEFG A Study Design B Data Collection Department of Chemistry, Illinois State University, Normal, IL, U.S.A. C Statistical Analysis D Data Interpretation Source of support: This work was supported in part by a grant from Sigma Xi: The Scientiﬁ c Research Society E Manuscript Preparation and by the Illinois State University Honors Program Undergraduate Research Scholarship (CLC) and NIH # 1 R15 F Literature Search GM/OD/52687-01A1 G Funds Collection

Summary

Background: The enzyme coproporphyrinogen oxidase (copro’gen oxidase) converts coproporphyrinogen-III (C-III) to protoporphyrinogen-IX via an intermediary monovinyl porphyrinogen. The A ring isomer coproporphyrinogen-IV (C-IV) has previously been shown to be a substrate for copro’gen oxidase derived from avian erythrocytes. In contrast to the authentic substrate (C-III) where only a small amount of the monovinyl intermediate is detected, C-IV gives rise to a monovinyl intermediate that accumulates before being converted to an isomer of protoporphyrinogen-IX. No kinetic studies have been carried out using the purifi ed human copro’gen oxidase to evaluate its ability to process both the authentic substrate as well as analogs. Materials/Methods: Therefore, purifi ed, cloned human copro’gen oxidase was incubated with C-III or C-IV at 37oC with various substrate concentrations (from 0.005 µM to 3.5 µM). The Km (an indication of molecular recognition) and Kcat (turnover number) values were determined. Results: The Km value for total product formation was about the same with either C-III or C-IV indicating the same molecular recognition. However, the catalytic effi ciency (Kcat/Km) of the enzyme for total product formation was not more than two fold higher using C-III relative to C-IV. Conclusions: Since the Km values are about the same for either substrate and the total Kcat/Km values are within two fold of each other, this could correlate with the increase of severity of porphyrias with monovinyl accumulation. The ability of the increased levels of C-IV to compete with the authentic substrate has important implications for clinical porphyrias. key words:EDUCATIONAL coproporphyrinogen oxidase • porphyria • Km • Kcat • substrate analog USE Full-text PDF: http://www.medscimonit.com/fulltxt.php?IDMAN=7723 Word count: 2471 Tables: 1 Figures: 5 References: 17

Author’s address: Marjorie A. Jones, Department of Chemistry, Illinois State University, Normal, IL 61790-4160, U.S.A., e-mail: [email protected]

BR420 Current Contents/Clinical Medicine • SCI Expanded • ISI Alerting System • Index Medicus/MEDLINE • EMBASE/Excerpta Medica • Chemical Abstracts • Index Copernicus

Electronic PDF security powered by ISL-science.com This copy is for educational use only - distribution prohibited. Med Sci Monit, 2005; 11(11): BR420-425 Cooper CL et al – An a ring isomer as substrate for coproporphyrinogen oxidase

BACKGROUND with the correct sequence of substituents for the second “B ring” propionate group to undergo oxidative decarboxyla- Porphyrias are a group of clinical disorders, either genet- tion. The Lash et al. paper also models the active site with ic or acquired, that are characterized by accumulation of C-IV leaving the active site and re-entering prior to the sec- BR one or more type of porphyrins in tissues as well as high- ond oxidative decarboxylation [11]. This isomer of the au- er levels of excretion in the urine and/or feces. These thentic substrate has the sequence of the methyl and pro- patients are generally characterized with mild to severe pionate groups on ring A switched, giving the molecule a mental retardation, photosensitivity, and problems in plane of symmetry that is absent in C-III, and also provid- the liver and/or bone marrow. Thus, understanding the ing two sequences of substituents that can be recognized at enzymes involved in the heme biosynthesis pathway and the active site. However, the active site model predicts that their ability to process both the authentic substrates and once the fi rst oxidative decarboxylation has occurred, the analogs of these substrates is important in helping to devel- second propionate side chain cannot be correctly orientat- op clinical therapies for porphyrias. Defects in the various ed until the intermediate has dissociated from the bind- enzymes of the synthetic pathway have been correlated with ing pocket, fl ipped over, and reentered into the active site. various classes of porphyrias but much work yet remains. This prediction provided a tentative explanation for the differences in the observed kinetics using both substrates Coproporphyrinogen oxidase (copro’gen oxidase; EC by Jackson et al. [4]. Since C-IV can be processed as a sub- 1.3.3.3) is the sixth enzyme in the biosynthetic pathway for strate by normal (wildtype) copro’gen oxidase, this would the production of heme. Coproporphyrinogen-III (C-III), result in a divinyl product with a concomitant accumula- the authentic substrate for the enzyme copro’gen oxidase, tion of a monovinyl species. We speculate that the accumu- undergoes two sequential oxidative decarboxylations at the lation of both the mono- and divinyl products from C-IV A and B ring propionates to produce fi rst a monovinyl prod- in the mitochondria could then be correlated with clinical uct and then a divinyl product (Figure 1A). symptoms of the patients with harderoporphyria, resulting from the defect in a previous enzyme rather than in the Harderoporphyria is a disorder often characterized by an copro’gen oxidase enzyme. However, the active site model accumulation of monovinyl porphyrins in the body and was designed from results observed using crude preparations considered to be related to mutations in the gene encod- of copro’gen oxidase from avian erythrocytes. As yet there ing copro’gen oxidase. In addition, C-III and its isomer co- have been no reports on the kinetics for C-IV metabolism proporphyrinogen-IV (C-IV) have been isolated from these using highly purifi ed human enzyme preparations. In this types of patients [1]. Yet, C-IV also has been reported in the work, experiments were performed using purifi ed prepara- urine of patients with a defect in the enzyme 5-aminolevulin- tions of the cloned human enzyme. The kinetic constants ic acid dehydratase [2]. Kühnel et al. [1] also reported the (Km, Kcat, and Kcat/Km) were determined using non-lin-

presence of C-IV in the urine of normal people, although ear regression under initial velocity (vo) conditions using in much lower amounts. either C-III or C-IV as substrate.

Modifi ed porphyrinogens have been used to probe the ac- MATERIAL AND METHODS tive site of copro’gen oxidase. C-IV was fi rst found to be a substrate for ox-liver copro’gen oxidase by Porra and Falk Coproporphyrin-IV tetramethyl ester was prepared by the in 1964 [3], and was subsequently shown to be converted cyclization of an a,c-biladiene intermediate following the to protoporphyrinogen-XIII via a monovinyl intermediate method reported by Lash et al. [10] and coproporphyrin (Figure 1B). Jackson et al., using crude enzyme preparations III tetramethyl ester was purchased from Aldrich Chemical from chicken red blood cells, reported that the monovinyl Company. Prior to reduction to the corresponding porphy- intermediate from C-IV accumulated to about 40% total rinogens, they were incubated overnight with 8.3 M HCl to porphyrinogen at intermediary incubation times, but was remove the methyl esters. eventually converted to the divinyl product [4]. This con- trasts to time study experiments with authentic C-III where Enzyme assay accumulationEDUCATIONAL of the monovinyl species, harderoporphyrin- USE ogen, was less than 10%. In addition, Buldain et al. showed Using the micro method of Jones et al. [12], either the C-III that incubations of C-IV with crude copro’gen oxidase prep- or C-IV porphyrinogen substrate (at a substrate concentra- arations from duck blood, chicken blood and beef liver gave tion of 1 µM) was incubated with copro’gen oxidase at times divinyl product at a rate of only 10% that of C-III [5]. These from 15 sec to 30 min. The fi nal reaction volume was 310 studies also showed that the type isomers coproporphyrin- µl which was made up of 300 µl of enzyme in 250 mM imi- ogen-I and coproporphyrinogen-II were not substrates for dazole buffer, pH 7.2 and 10 µl of substrate. In another set copro’gen oxidase. On the bases of these and other inves- of experiments, substrate concentration was varied from 0 tigations with substrate analogs [6–9], Lash et al. proposed to 3.5 µM while holding the incubation time constant at 15 a substrate recognition model for the enzyme [10]. In this sec. For all reactions, the temperature was 37oC and the en- model, there are requirements for the correct sequence of zyme was 7.5 µg (0.20 nmoles) per incubate. the peripheral substituents around the macrocycle. In or- der to provide the correct “fi t”, the sequence R Me-P Me-P The reactions were stopped by addition of 3/7 (v/v) acetic

is required, where Me= –CH3, P= –CH2CH2CO2H and R is a acid/ethyl acetate, followed by extraction and Fischer esteri- small nonpolar grouping such as Me, ethyl, or vinyl. When ﬁ cation overnight. In all experiments, a zero incubation time C-III is metabolized, a vinyl group is generated at ring A. control (addition of acetic acid/ethyl acetate before addi- The model predicts that the substrate remains in the active tion of substrate) was performed for comparison. Following site and can rotate through 90o and present the active site neutralization and extraction of the porphyrin methyl esters, BR421

Electronic PDF security powered by ISL-science.com This copy is for educational use only - distribution prohibited. Basic Research Med Sci Monit, 2005; 11(11): BR420-425

EDUCATIONAL USE

Figure 1. (A) Oxidative decarboxylation of C-III to P-IX via harderoporphyrinogen. (B) Oxidative decarboxylation of C-IV to P-XIII via a monovinyl intermediate.

they were evaluated using normal phase High Performance was used with 35/65 (v/v) ethyl acetate/cyclohexane at a Liquid Chromatography (HPLC; Beckman System Gold). A ﬂ ow rate of 1.3 ml per minute; eluates were evaluated spec- normal phase column (Beckman Silica 5 µ, 4.6 mm × 25 cm) trophotometrically at a wavelength of 404 nm. Data were BR422

A BR

B Figure 3. SDS-PAGE gel to show the purity of the copro’gen oxidase. In lane 1 are MW standards (97, 66, 45, 31, and 21 kDa), lanes 2 and 3 are the supernatant (1 and 10 µl), lane 4 is the Ni2+ column ﬂ ow through (10 µl), and lanes 5 and 6 are the elution fractions (10 and 2 µl) containing the puriﬁ ed enzyme.

at 35,000 × g for 30 minutes to pellet out the insoluble protein and membranes. The enzyme, which has the 6 his tag, was isolated by the Ni2+ affi nity procedure of Medlock and Dailey [13]. Protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using the Figure 2. (A) Representative HPLC Chromatogram for the methyl Laemmli method [14] to test the purity and apparent mo- esters of the porphyrin products resulting from incubations lecular weight of the enzyme. The Bradford Protein Assay of C-III with pure copro’gen oxidase enzyme. (B) HPLC [15] was used to evaluate the concentration of the enzyme. Chromatogram for incubations with C-IV. Zero time Bovine Serum Albumin (BSA) was used as the standard. (unreacted) peaks shown with a dotted line and porphyrin products isolated after incubation with enzyme with a RESULTS solid line. Peak A corresponds to methylene chloride (used to dissolve the sample), peak B to protoporphyrin (divinyl Highly purifi ed enzyme (30 mg/liter of cell culture) was ob- product), peak C to harderoporphyrin (monovinyl product), tained and only a single band of MW 37,700 Da was observed and peak D to C-III or C-IV (unreacted substrates) in Figure by SDS-PAGE (Figure 3). Figure 4A shows the time depend- 2A and 2B, respectively. ent accumulation of divinyl, monovinyl, and total products after incubation of 7.5 µg purifi ed enzyme with the authentic C-III substrate (1 µM). Total product was calculated as the sum of the monovinyl and divinyl products. Figure 4B analyzed using the Gold Nouveau Software and reported shows the time dependent accumulation of divinyl, monovi- as% product accumulatedEDUCATIONAL or subsequently converted to the nyl, and total products after incubation of USEC-IV under the units of Medlock & Dailey [13]. Representative chromato- same conditions. These data are the mean of three separate grams are shown in Figure 2. Figure 2A shows product for- experiments. The range of high and low values about the mation after zero or 20 minutes of incubation of C-III with mean value was 5%. The data for the apparent linear por-

enzyme. Figure 2B shows product formation after zero or tion of the lines (vo; % product/min) were evaluated by lin- 20 minutes of incubation of C-IV with copro’gen oxidase. ear regression analysis. The initial velocity (vo) for monovi- In both cases the unreacted substrate, the monovinyl, and nyl product accumulation is about 50 times faster (vo=106 the divinyl products are well resolved. relative to 2.4) with C-IV than with C-III. The initial veloci-

ty (vo) for divinyl product accumulation is about the same Isolation and puriﬁ cation of copro’gen oxidase (vo=28 relative to 35) with either substrate. C-IV total product accumulation reached an asymptote three to four fold

The human cloned enzyme was grown in E. coli strain faster relative to C-III (vo=139 relative to 37). Using either BL21(DE3)RIL (Stratagene) overnight at 37°C, with shak- substrate, approximately the same ﬁ nal total product accu- ing at 250 rpm, in one-liter cultures using a medium con- mulation was evident. sisting of 10 g tryptone, 5 g yeast extract, 10 g NaCl, and

100 mg ampicillin. Cells were isolated by centrifugation Figure 5A shows vo (nmole product/min/pmole enzyme) (12,000 × g, 10 min) and lysed using the French hydraulic as a function of substrate over a concentration range of

pressure cell (20,000 psi). Then the lysate was centrifuged 0.005 to 3.5 µM using C-III. Figure 5B shows vo as a func- BR423

Electronic PDF security powered by ISL-science.com This copy is for educational use only - distribution prohibited. Basic Research Med Sci Monit, 2005; 11(11): BR420-425

A B

Figure 4. (A) Time course study with C-III as the substrate showing the time dependency of product accumulation. (B) Time course with C-IV showing the time dependency of product accumulation. Triangles represent total product, squares show divinyl product, and circles indicate of monovinyl product intermediate accumulation. All values were the mean of three separate experiments. The range of high and low values about the mean was 5%.

A B

Figure 5. Eﬀ ect of substrate concentration on initial velocity (vo). (A) Substrate concentration curve with C-III as substrate. (B) Substrate concentration curve with C-IV as substrate. Triangles represent total product, circles show divinyl product, and squares indicate monovinyl product intermediate accumulation. The range of high and low values about the mean value of three separate experiments was 5%.

Table 1. Kinetic constants of copro’gen oxidase for monovinyl, divinyl, and total product accumulated.

Substrate Km (µM) Kcat (min-1) Kcat/Km C-III 0.13 0.20 1.50 Monovinyl Product EDUCATIONALC-IV 0.65 1.70 USE 2.60 C-III 0.70 3.30 4.70 Divinyl Product C-IV 0.83 0.70 0.85 C-III 0.68 3.60 5.30 Total Product C-IV 0.78 2.60 3.30

tion of substrate over a concentration range of 0.005 to using C-IV relative to C-III. For the divinyl product ac- 3.5 µM using C-IV. The kinetic constants (Km, Kcat, and cumulated, there was essentially no difference in Km us- Kcat/Km), using either substrate, are shown in Table 1. We ing either substrate, but almost a 5 fold increase in Kcat used the Michaelis-Menton assumptions and linear trans- using C-III relative to C-IV. The total product accumula- formation of the data to obtain the kinetic constants, Km tion (the sum of the mono and divinyl products detected) and Kcat. For the production of monovinyl product there shows about the same Km for either substrate, and a 1.5 is a 5 fold increase in Km when using C-IV as the substrate fold difference in Kcat values with C-III giving the higher relative to C-III, but more than an 8 fold increase in Kcat turnover number. BR424

DISCUSSION authors speculated about which amino acids were involved in the active site, this enzyme structure was solved in the absence Using purifi ed wildtype human copro’gen oxidase, the re- of substrate. This is likely due to the diffi cultly of keeping the sults clearly demonstrate that C-IV is a good substrate for this substrate in the reduced form recognized by the enzyme dur- BR enzyme and binds in the active site with affi nity comparable ing crystallization. It is not clear whether the active site has yet to the normal pathway C-III substrate. This is not surprising been correctly identifi ed. Thus, the factors infl uencing sub- since C-IV can fi t in either of two orientations that arise from strate selectivity are still not completely understood. its plane of symmetry. Our data indicate that C-IV is a better substrate for human enzyme than that of the other species Acknowledgements tested by Buldain et al. [5]. Jackson et al. [4] reported that the monovinyl product from C-IV accumulates to about 40% The cDNA encoding the 6x-histidine-tagged human at intermediary times, but was eventually converted to divinyl copro’gen oxidase was a generous gift from Dr. H.A. Dailey product. Our data show that the monovinyl product accumu- of the University of Georgia (Athens, GA). lates to 45% but was not apparently converted 100% into divinyl product with the human purifi ed enzyme, even up to REFERENCES: 30 minutes of incubation. For divinyl product accumulation, C-III results in a higher turnover (larger Kcat) and more cat- 1. Kühnel A, Fross U, Jacob K, Doss MO: Studies on Coproporphyrin alytic effi ciency (larger Kcat/Km) with about the same bind- Isomers in Urine and Feces in the Porphyrias. Clin Chim Acta, 1999; ing affi nity (Km) relative to C-IV. When considering the to- 282: 45–58 2. Jacob K, Doss MO: Composition of Urinary Coproporphyrin tal product accumulation, both of the substrates are equally Isomers I-IV in Human Porphyrias. Euro J Clin Chem Clin Biochem, processed under vo conditions. From the literature, a range 1993; 31: 617–24 of Km values between 0.05 and 47 µM for C-III have been re- 3. Porra RJ, Falk JE: The Enzymatic Conversion of Copropor phyr- ported for this enzyme [13]. The Km values determined here inogen III into Protoporphyrinogen IX. Biochem J, 1964; 90: 69–75 fall within the literature range. Our Km values were obtained 4. Jackson AH, Elder GH, Smith SG: The Metabolism of Copropor- using highly purifi ed enzyme at a very short incubation time, phyrinogen-III into Protoporphyrin-IX. Int J Biochem, 1978; 9: 877–82 while previous studies were conducted using a variety of en- 5. Buldain G, Hurst J, Frydman RB, Frydman B: Synthesis of the Tricarboxylic Porphyrin Enzymically Formed from Coproporphyrinogen zyme sources, enzyme purities, incubation times, and assay IV. J Org Chem, 1977; 42: 2953–56 procedures which may explain the vast range of values. 6. Al-Hazimi HMG, Jackson AH, Knight DW, Lash TD: Synthetic and Biosynthetic Studies of Porphyrins. Part 7. The Action of When the total product accumulation of C-III was compared Coproporphyrinogen Oxidase of Coproporphyrinogen-IV: Synthesis of Protoporphyrin-XIII, Mesoporphyrin-XIII, and Related Tricarboxylic with C-IV as a function of time, the apparent initial velocity us- Porphyrins. J Chem Soc, Perkin Trans, 1987; 1: 265–76 ing C-IV was about 3 fold higher than when using C-III. Thus, 7. Battersby AR, Hamilton AD, McDonald E et al: Biosynthesis the position of the propionate group on the A ring leads to dif- of Porphyrins and Related Macrocycles. Part 13. Structure of the ferent kinetics for the fi rst oxidative decarboxylation for both Protoporphyrin Isomer Derived from Coproporphyrinogen-IV substrates. Different kinetics are also evident for the second by Reaction of Beef-Liver Coproporphyrinogenase: Synthesis of Protoporphyrin-XIII. J Chem Soc, Perkin Trans, 1980; 1: 1283 oxidative decarboxylation with the accumulation of the divinyl 8. Lash TD, Hall T, Mani UN, Jones MA: Normal and Abnormal product being much lower when using C-IV; C-IV resulted in a Heme Biosynthesis. 3. Synthesis and Metabolism of Tripropionate 5.5 fold lower Kcat/Km value relative to the C-III data. Analogues of Coproporphyrinogen-III: Novel Probes for the Active Site of Coproporphyrinogen Oxidase. J Org Chem, 2001; 66: 3753–59 CONCLUSIONS 9. Lash TD, Keck ASIM, Mani UN, Jones MA: Unprecedented Overmetabolism of a Porphyrinogen Substrate by Coproporphyrinogen Oxidase. Bioorg Med Chem Lett, 2002; 12l: 1079–82 These data support the model that there is a limiting step in 10. Lash TD, Mani UN, Drinan MA et al: Normal and Abnormal the second oxidative decarboxylation of C-IV. Thus our mod- Heme Biosynthesis. 1. Synthesis and Metabolism of Di- and el, which predicts that C-IV comes completely out of the ac- Monocarboxylic Porphyrinogens Related to Coproporphyrinogen- III and Harderoporphyrinogen: A Model for the Active Site of tive site and fl ips over prior to re-entering the active site lead- Coproporphyrinogen Oxidase. J Org Chem, 1999; 64: 464–77 ing to divinyl product formation, is reasonable for the human 11. Phillips JD, Whitby FG, Warby CA et al: Crystal Structure of the Oxygen- form of the enzyme.EDUCATIONAL This then would allow either C-III or C- dependant Coproporphyrinogen Oxidase (Hem13p) USE of Saccharomyces IV to compete with the monovinyl intermediate for the active cerevisiae. J Biol Chem, 2004; 279: 38960–68 site, thus lowering the divinyl product production from either 12. Jones MA, Thientanavanich P, Anderson MD, Lash TD: Comparison of porphyrinogen. In vivo, this would increase the time that these Two Assay Methods for Activities of Uroporphyrinogen Decarboxylase and Coproporphyrinogen Oxidase. J Biochem Biophys, 2003; 55: 241–49 porphyrinogens reside in the cell especially in the mitochon- 13. Medlock AE, Dailey HA: Human Coproporphyrinogen Oxidase is Not drial compartment where copro’gen oxidase is located [16], a Metalloprotein. J Biol Chem, 1996; 271: 32507–10 allowing them to spontaneously oxidize to the porphyrin form 14. Laemmli UK: Cleavage of Structural Proteins During Assembly of the not utilized by copro’gen oxidase. This could, therefore, in- Head of Bacteriophage T4. Nature, 1970; 227: 680–85 crease the severity of the porphyria. Thus detection of monovi- 15. Bradford MM: A Rapid and Sensitive Method for the Quantitation of nyl porphyrins in urine need not only be related to defects in Microgram Quantities of Protein Utilizing the Principle of Protein-Dye the copro’gen oxidase enzyme. Our data do not support or re- Binding. Anal Biochem, 1976; 72: 248–54 ject any of the proposed mechanisms for the oxidative decar- 16. Grandchamp B, Phung N, Nordmann Y: The Mitochondrial Localization of Coproporphyrinogen III Oxidase. Biochem J, 1978; 176: 97–102 boxylations performed by this enzyme [17]. The active site of 17. Akhtar M: The Modifi cation of Acetate and Propionate Side Chains the enzyme has not yet been well established although several During the Biosynthesis of Haem and Chlorophylls: Mechanistic crystal structures have been published [11]. An X-ray crystal- and Stereochemical Studies. In: The Biosynthesis of the Tetrapyrrole lographic structure for copro’gen oxidase has been recently Pigments. Chichester (Ciba Foundation Symposium 180). John Wiley & Sons, 1994; 131–55 solved for the Saccharomyces cerevisiae oxygen dependant form of the enzyme, but not the human form [11]. Although the BR425

Electronic PDF security powered by ISL-science.com This copy is for educational use only - distribution prohibited. Index Copernicus Global Scientific Information Systems for Scientists by Scientists

www.IndexCopernicus.com

EVALUATION & BENCHMARKING PROFILED INFORMATION NETWORKING & COOPERATION VIRTUAL RESEARCH GROUPS GRANTS PATENTS CLINICAL TRIALS JOBSJOBS STRATEGIC & FINANCIAL DECISIONS

IndexIndex IC Scientists IC Virtual Research Groups [VRG] Effective search tool for Web-based complete research Copernicus collaborators worldwide. environment which enables researchers CopernicusCopernicus Provides easy global to work on one project from distant networking for scientists. locations. VRG provides: C.V.'s and dossiers on selected integratesintegrates customizable and individually EDUCATIONALscientists available. Increase USE self-tailored electronic research your professional visibility. protocols and data capture tools, IC Journal Master List IC Patents statistical analysis and report creation tools, Scientific literature database, Provides information on patent profiled information on literature, including abstracts, full text, registration process, patent offices publications, grants and patents and journal ranking. and other legal issues. Provides related to the research project, Instructions for authors links to companies that may want available from selected journals. to license or purchase a patent. administration tools.

IC Grant Awareness IC Lab & Clinical Trial Register IC Conferences Need grant assistance? Step-by-step information on Provides list of on-going laboratory Effective search tool for how to apply for a grant. Provides or clinical trials, including worldwide medical conferences a list of grant institutions and research summaries and calls for and local meetings. their requirements. co-investigators.

Electronic PDF security powered by ISL-science.com This copy is for educational use only - distribution prohibited.